Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding

Abstract

Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane.IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.

Keywords: Dot/Icm system; Legionella pneumophila; cryoelectron tomography; effector proteins; nanomachine; protein transport; secretion system; type IV secretion; whole-cell tomography.

FIG 1

Localization pattern of individual L. pneumophila T4SS machines. (A to C) Central sections of tomograms showing entire L. pneumophila cells at different stages of bacterial growth: stationary, elongation, and division phase. Panels are arranged in a progressing order of bacterial growth. Outer membrane (OM), inner membrane (IM), and granule (P) are annotated. Dotted circles indicate the T4SS machines. (D to F) 3D renderings of the tomograms shown in panels A to C. The upper right corner insets represent magnified views of boxed regions.

FIG 2

Unique structural intermediates suggest the “outside-inside” model of the L. pneumophila T4SS machine assembly. (A to E) Central sections of the class average structures showing distinct structural conformations. Panels are arranged in an order of increasing structural complexity. (F to O) 3D surface models of structural intermediates in panels A to E in side views (F to J) and bottom views (K to O).

FIG 3

Docking of the DotB ATPase complex induces conformational change of the entire cytoplasmic complex. (A and G) Class average structures shown in Fig. 2D and E. (B and H) Central sections of asymmetrically reconstructed cytoplasmic complex structures of the DotB-free (B) and DotB-bound (H) intermediates. (C and I) Cross-section views at the positions indicated in panels B and H. (D to F) 3D renderings of the DotB-free cytoplasmic complex. (J to L) 3D renderings of the DotB-bound cytoplasmic complex. (M to R) Superimposition of the DotB-free and DotB-bound cytoplasmic complex.

FIG 4

DotB-induced conformational change opens the type IV secretion channel. (A and E) Central sections of cytoplasmic complex structures of the DotB-free (A) and DotB-bound (E) intermediates. Blue asterisks highlight the location of the cytoplasmic end of the secretion channel. (B to D and F to H) 3D renderings of the averaged structure shown in panels A and D, respectively. Twofold symmetry was imposed along the axis of innate 2-fold pseudosymmetry to facilitate 3D segmentation. PDB entry 6GEB was docked in the place of DotB density. (I) Proposed model of L. pneumophila T4SS activation. Docking of DotB complex induces conformational changes that open the IM secretion channel for effector translocation.